Production of frambinone by a recombinant fungal microorganism

ABSTRACT

The invention relates to a genetically modified fungal microorganism for the production of frambinone, the microorganism having the following characteristics: —the capacity to produce frambinone from tyrosine; and —a limited capacity or no capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate; and to the use of same for producing frambinone.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of U.S. patent application Ser. No. 16/302,527, filed Nov. 16, 2018, which is a U.S. national phase pursuant to 35 U.S.C. § 371 of International Patent Application No. PCT/FR2017/051407, filed Jun. 2, 2017, which claims priority to French Application No. 1655089, filed Jun. 3, 2016, the disclosures of which are all herein incorporated by reference in their entireties.

This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (filename: 53662A_Seqlisting.txt; Size: 43,436 bytes; Created: Aug. 27, 2020), which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods for effectively producing frambinone from tyrosine using fungal microorganisms and suitable culture conditions.

DESCRIPTION OF THE PRIOR ART

Raspberry flavor (Rubus idaeus) is linked to more than 200 compounds, but frambinone, a natural phenolic compound, is the compound with the greatest impact, defining its characteristic taste (Klesk et al., 2004, J. Agric. Food Chem. 52, 5155-61; Larsen et al., 1991, Acta Agric. Scand. 41, 447-54). It can also be found in other fruits and vegetables, including peaches, apples and rhubarb (Beekwilder et al., 2007, Biotechnol. J. 2, 1270-79). As it is present only in small amounts in raspberries (1-4 mg per kg of fruit), natural frambinone is highly valuable (Larsen et al., 1991). Because it has limited natural availability, producing it by means of biotechnology is highly desirable.

Frambinone (CAS no: 5471-51-2), also called raspberry ketone or 4-(4-hydroxyphenyl)butane-2-one, has the following structure:

Frambinone can be used in many applications, notably in the food business and for cosmetics (as fragrance), for agriculture (as bait/trap for insects), in the health industry (as a weight-loss product) or in medicine (as a melanogenisis inhibitor).

Frambinone can be obtained from a 4-step biosynthesis, starting with aromatic amino acid L-tyrosine as substrate (FIG. 1: Beekwilder et al., 2007, Biotechnol. J. 2, 1270-79): Tyrosine is deaminated by a tyrosine ammonia-lyase TAL, EC 4.3.1.23) to form coumaric acid. Catalyzed by a 4-coumerate:CoA ligase (4CL, EC 6.2.1.12), a Coenzyme A (CoA) molecule is grafted onto coumaric acid. The coumaroyl-CoA is then converted by a benzalacetone synthase (BAS, EC 2.3.1.212) into 4-hydroxybenzalacetone. This reaction is a decarboxylating condensation and uses a malonyl-CoA unit as co-substrate. The final step is reduction of the 4-hydroxybenzalacetone to frambinone by a benzalacetone reductase (BAR EC 1.3.1.x).

An alternative substrate for the production of frambinone is coumaric acid, an intermediate in the path described above (denoted (2) in FIG. 1) However, in light of the price of the substrates used for bioconversion, industrial applications are only profitable when tyrosine is used rather than coumaric acid.

L-phenylalanine (denoted (7) in FIG. 1) can also be used as a substrate as it can be converted into coumaric acid via cinnamic acid (denoted (8) in FIG. 1) In fact, formation of frambinone in plants uses a general phenylpropanoids path, which starts with phenylalanine (Borejsza-Wysocki and Hrazdina, 1994, Phytochemistry 35, 623-28.). The first step is deamination catalyzed by a phenylalanine ammonia lyase (PAL. EC 4.3.1.24) The cinnamic acid produced is then hydroxylated by cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) to form coumaric acid, which is converted as described above. C4H is a P450 cytochrome and is linked to the endoplasmic reticulum membrane. Its expression seems problematic and full activity also requires an additional enzyme (cytochrome P450 reductase, CPR) (Bassard et al., 2012, Plant Cell 24, 4465-82; Schückel et al., 2012, ChemBioChem 13, 2758-63; Winkel, 2004, Rev. Plant Biol. 55, 85-107).

Biosynthesis of two other compounds of biotechnological interest, resveratrol and naringenin, show many similarities to the path proposed for frambinone (Jeandet et al., 2012, J. Biomed. Biotechnol. 2012, 1-14; Lussier et al., 2012, Comput. Struct. Biotechnol. J. 3, 1-11.). TAL and 4CL are used to convert tyrosine and coumaric acid into coumaroyl-CoA. A stilbene synthase (STS) or chalcone synthase (CHS) then catalyzes the consecutive condensation with three malonyl-CoA units to form resveratrol or chalcone naringenin, respectively.

In light of the interest in an alternative production to the chemical synthesis of frambinone, transposing these ways of synthesizing recombinant microorganisms has been attempted, in particular in Escherichia coli and Saccharomyces cerevisiae.

GB 2 416 769 in particular describes the possibility of producing frambinone using a microorganism (particularly bacteria and yeast) containing a 4CL coding sequence and BAS, at least one from heterologous source. It can additionally include a BAR, C4H, PAL and/or CHS coding sequence, the BAR coding sequence being advantageously endogenic. In the examples, this document reports:

-   -   cloning the raspberry gene CHS;     -   cloning the tobacco gene 4CL;     -   cloning the rhubarb gene BAS;     -   the transformation of E. coli with the raspberry gene BAR;     -   the production of benzalacetone and frambinone (0.2 μg in 50 mL)         from coumaric acid in E. coli transformed with BAS and 4CL;     -   the production of frambinone from benzalacetone in E. coli         having endogenous BAR activity.

Based on the assumption that CHS has BAS activity, GB 2 416 770 describes the possibility of producing benzalacetone and frambinone with a microorganism comprising a 4CL coding sequence (tobacco, for example) and CHS (raspberry bush or petunia), at least one being a heterologous source. It can additionally include a BAR coding sequence (raspberries for example), C4H and PAL. In the examples, this document reports:

-   -   the production of benzalacetone and naringenin from coumaric         acid in E. coli transformed with CHS and 4CL;     -   the building of a mutated protein CHS (CHS*) presumed to have         higher BAS activity.     -   the production of benzalacetone and frambinone (14.2/0.3 μg in         50 mL) from coumaric acid in E. coli with endogenous BAR         activity and transformed with CHS/CHS* and 4CL;     -   the production of benzalacetone from coumaric acid in S.         cerevisiae (with endogenous BAR activity) transformed with CHS         and 4CL. No value is given in connection with frambinone.         Furthermore, the mutant protein CHS* seems ineffective.

Similarly, Beekwilder et al. (2007, Biotechnol. J. 2, 1270-79) only report the successful production of frambinone from coumaric acid in E. coli with CHS and 4CL but with a yield of 0.3 mg/L. There has been no convincing data reported for production with yeast.

Recently, Lee et al. (2016, Microb. Cell Factories 15. doi:10.1186/s12934-016-0446-2) have demonstrated the synthesis of frambinone from coumaric acid by expressing a 4CL gene and BAS in S. cerevisiae (up to 8 mg/L). Furthermore, the expression of PAL/TAL and C4H made de novo production of frambinone possible (up to 4 mg/L).

Moreover, Rodriguez et al. (2015, Metabolic Engineering 31, 181-188) described a genetically modified yeast for the production of coumaric acid, the precursor of flavonoids like resveratrol and naringenin. So it is reported that inactivation of genes PDC5 and ARO10 coding a pyruvate decarboxylase and a phenylpyruvate decarboxylase, respectively, leads to overproduction of coumaric acid.

There is, then, an obvious need to develop new technical solutions offering effective production of frambinone.

DESCRIPTION OF THE INVENTION Definitions

The definitions below correspond to the meaning generally used in the context of the invention and should be taken into account, unless another definition is explicitly indicated.

Within the meaning of the invention, the articles “a” and “an” are used to refer to one or more (for example, at least one) units of the grammatical subject of the article. As an example, “an element” refers to at least one element, i.e., one or more elements.

The terms “about” or “approximately”, used in reference to a measurable value such as a quantity, a duration, and other similar values, must be understood as encompassing measurement uncertainties of ±20% or ±10%, preferably ±5%, still more preferably ±1%, and particularly preferably ±0.1% of the specified value.

Intervals: throughout the present description, the various features of the invention may be presented in the form of intervals of values. It must be understood that the description of values in the form of intervals is intended solely to make reading easier and must not be interpreted as a rigid limitation of the scope of the invention. As a result, the description of an interval of values should be considered as specifically disclosing all of the possible intermediate intervals as well as each of the values within this interval. For example, the description of an interval from 1 to 6 should be considered as specifically describing each of the intervals that it comprises, such as the intervals from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each of the values in this interval, for example 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This definition is valid independently of the scope of the interval.

The term “isolated” must be understood in the context of the invention as being synonymous with withdrawn or removed from its natural environment or state. For example, an isolated nucleic acid or peptide is a nucleic acid or peptide taken out of the natural environment in which it is typically found, whether it involves a plant or a living animal, for example. Thus, a nucleic acid or a peptide naturally present in a living animal is not an isolated nucleic acid or peptide within the meaning of the present invention, while the same nucleic acid or peptide, partially or completely separated from the other elements present in its natural context, is in turn “isolated” within the meaning of the invention. An isolated nucleic acid or peptide may exist in a substantially purified form, or may exist in a non-native environment, for example a host cell.

In the context of the invention, the following abbreviations are used for the most common nucleic acid bases. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

Unless otherwise indicated, within the meaning of the invention, a “sequence of nucleotides coding for a sequence of amino acids” refers to all of the nucleotide sequences that code for the amino acid sequence, including the degenerated nucleotide sequences making it possible to obtain said sequence of amino acids. The nucleotide sequence that codes for a protein or an RNA or a cDNA may optionally comprise introns.

The terms “coding” or “coding for”, “code” or “code for” refer to the property inherent to the specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA or a mRNA, to serve as a matrix for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (for example rRNA, tRNA and mRNA), or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein if the transcription and the translation of the mRNA corresponding to this gene produce the protein in a cell or another biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is generally described in the listings of sequences and databases, and the non-coding strand, used as matrix for the transcription of a gene or cDNA, can be designated as coding for the protein or another product of this gene or cDNA.

The term “polynucleotide” as used in the context of the invention is defined as a chain of nucleotides. Furthermore, the nucleic acids are nucleotide polymers. Thus, the terms nucleic acids and polynucleotides as used in the scope of the invention are interchangeable. It is well known in the field of molecular biology and genetic engineering that nucleic acids are polynucleotides, which can be hydrolyzed into monomers. Nucleotides in monomer form can be hydrolyzed into nucleosides. As used in the context of the invention, the term polynucleotide refers, non-limitingly, to any type of nucleic acid molecules, i.e., where the nucleic acid molecules can be obtained by any means available in the art, including by recombinant means, namely the cloning of sequences of nucleic acids from a recombinant library or the genome of a cell, by using ordinary cloning technologies such as PCR, or by synthesis.

Within the meaning of the invention, the terms “peptide”, “polypeptide” and “protein” are used interchangeably and refer to a compound made up of amino acid residues covalently bonded by peptide bonds. By definition, a protein contains at least two amino acids, without limitation regarding the maximum number of amino acids. Polypeptides indifferently comprise several peptides and/or proteins, which in turn comprise two or more amino acids linked to one another by peptide bonds. As used here, the term refers both to short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, which are generally referred to in the art as proteins, many types of which exist. “Polypeptides” for example comprise biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others. Polypeptides comprise natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The terms “homologous” and “identical” refer to the similarity of sequence or identity of sequence between two polypeptides or between two nucleic acid molecules. When a position in each of the two compared sequences is occupied by the same amino acid monomer base or sub-unit (for example, when a position in each of the two DNA molecules is occupied by an adenine), then the molecules are homologous or identical for this position. The percentage of identity between two sequences depends on the number of corresponding positions shared by the two sequences, and corresponds to this number divided by the number of positions compared and multiplied by 100. For example, if 6 out of 10 of the positions in two paired sequences are identical, then the two sequences are 60% identical. As a general rule, the comparison is done by aligning the two sequences so as to provide maximal homology/identity.

A “vector” within the meaning of the invention is a molecular construct that comprises an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the inside of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term “vector” for example refers to a plasmid with autonomous replication or a virus. The term must also be interpreted as comprising non-plasmid or non-viral compounds that facilitate the transfer of nucleic acids into the cells, for example compounds of polylysine, liposomes, and the like.

The term “expression vector” refers to a vector comprising a recombinant polynucleotide, which comprises expression control sequences operationally linked to a nucleotide sequence to be expressed. An expression vector in particular comprises cis-acting expression elements; other elements for the expression can be provided by the host cell or by an in vitro expression system. The expression vectors within the meaning of the invention include all those known in the art, such as cosmids, plasmids (for example naked or contained in liposomes) and viruses (for example lentiviruses, retroviruses, adenoviruses and adeno-associated viruses), which incorporate the recombinant polynucleotide.

The term “promoter” as used here is defined as a DNA sequence recognized by the synthesis machinery of the cell, or the introduced synthesis machinery, necessary to initiate the specific transcription of a sequence of polynucleotides.

Within the meaning of the invention, the terms “promoter/regulator sequence” refer to a nucleic acid sequence, necessary for the expression of the polynucleotide linked operationally to the promoter/regulator sequence. In some cases, this sequence may be the base sequence of the promoter, while in other cases, this sequence may also comprise an activator sequence and other regulator elements, useful for the expression of the polynucleotide. The promoter/regulator sequence may for example be a sequence allowing the expression of the polynucleotide that is specific to a tissue, i.e., preferably being produced in that tissue.

Within the meaning of the invention, a “constitutive” promoter is a nucleotide sequence which, when operationally linked to a polynucleotide, leads to an expression of the polynucleotide under most or all of the physiological conditions of the cell.

Within the meaning of the invention, an “inducible” promoter is a nucleotide sequence which, when operationally linked to a polynucleotide, leads to an expression of the polynucleotide only when an inducer of the promoter is present in the cell.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a genetically modified fungal microorganism for the production of frambinone, said microorganism having the following characteristics:

-   -   a capacity to produce frambinone from tryosine; and     -   a limited capacity or no capacity to break tyrosine down into         tyrosol, p-hydroxyphenylacetaldehyde and/or         p-hydroxyphenylacetate.

Within the scope of the invention, the expression “genetically modified microorganism” means that the microorganism according to the invention is not found in nature and is modified by introducing new genetic elements and/or deleting or modifying endogenous genetic elements of the microorganism. Such a microorganism can be subject to selection pressure, combining directed mutagenesis and being cultured in the selection medium.

In what follows and for reasons of simplification, the term “genetic element” is equivalent to “gene” or “sequence”. A nucleic acid sequence, then, can have any type of functionality. For example, it could be a coding sequence (coding specifically for an enzyme from the synthesis or degradation pathway of interest), or a regulatory sequence, particularly a promoter or a terminator. Especially when a coding sequence is concerned, it is optimizable, that is, modifiable to integrate the preferred codons of the host, here a fungal microorganism, wherein this sequence is expressed. According to another preferred embodiment, only the coding sequence in the gene of interest, or ORF (“Open Reading Frame”) is isolated and implemented.

According to an initial phase, the new genetic elements introduced in the microorganism target of the invention are genetic elements called exogenous or heterologous elements, which can also be synthetic or come from other organisms (or sources). In particular, a microorganism can express exogenous or heterologous genes if they are introduced into the aforesaid microorganism with all the elements needed to express in the host microorganism.

According to another embodiment, the endogenous genes can be modified to modulate their expression and/or their activity, for example by introducing mutations into their coding sequence to modify the genetic product or modify the regulating sequences, for example, further introducing heterologous sequences or replacing endogenous regulating sequences. Modulating the endogenous genes can result in over-expression or increased activity of the endogenous gene produced or, on the other hand, decreased expression or activity.

What is more, supernumerary or additional copies of an endogenous gene can also be introduced into the microorganism thereby increasing the level of expression and thus the activity of the product coded by the gene.

The techniques used to introduce DNA into a host (or transformation) are well known to the person skilled in the art and include permeabilization of the membranes by applying an electric field (electroporation) with heat (application of a thermal shock) or chemically, using lithium acetate, for instance.

The genetic elements introduced can be integrated into the genome of the host, notably by homologous recombination or chromosomal integration, advantageously with the use of integrative cassettes, or expressed extrachromosomically assisted by plasmids or vectors. Different types of plasmids, advantageously self-replicating, are well known by the person skilled in the art, differing notably by the origin of replication, the promoter (inducible or constitutive), the marker (for instance resistance to antibiotics or capacity to grow in a selective medium) and the number of copies per cell.

Advantageously in a fungal microorganism, genetic chromosomal integration, notably of expression cassettes bearing heterologous genes or supernumerary copies of endogenous genes, is done using the technique called “modular cassette integration technique.” According to a particular embodiment, the gene(s) are integrated at the HO locus. In the event of the integration of several cassettes, they are chosen for having extremities with homologous sequences, called recombination regions (RR) permitting homologous recombination and integration in the order desired and the position desired for different cassettes. Advantageously, one of the cassettes called “cassette marker” codes a marker, for example resistance to an antibiotic or capacity to grow in a selective medium, making it possible to select or identify microorganisms in which chromosomal integration has taken place. Advantageously, an expression cassette comprises the coding part or ORF of a gene of interest, notably the enzymes involved in the biosynthetic path to frambinone from tyrosine, placed under control of regulator sequences, advantageously at least one promoter and one terminator, which can be the native regulator sequences of this gene or heterologous sequences chosen for their functionality and/or effectiveness in a host microorganism.

Inactivation of the endogenous genes can be done for instance by introducing by homologous recombination at the target gene level, a cassette, either at the regulator regions level, thus inhibiting gene expression, or the level of sequence coding resulting in inactivation of the gene produced. According to a particular embodiment, the cassette is a cassette marker, advantageously comprising a dominant gene marker controlled by a promoter and a terminator. Even more advantageously, said cassette comprises at its 5′ and 3′ extremities homologous regions in the 5′ and 3′ regions of the targeted gene, for example, corresponding to 5′ in the promoter of the targeted gene and corresponding to 3′ in the terminator of the targeted gene. The cassette can also contain loxP sites making it possible to excise said cassette from the genome due to the action of cre recombinase.

Within the scope of the invention, adapted markers are the genes conferring resistance to antibiotics that are then introduced into the culture medium of the genetically modified microorganism to ensure selection and maintenance of the genetic modification. Numerous markers are available to the person skilled in the art, for example:

-   -   gene kanMX4 conferring resistance to geneticin (or G418);     -   gene hphNT1 conferring resistance to hygromycin B;     -   gene bsd conferring resistance to blasticidin;     -   gene ble conferring resistance to phleomycin.

A good introduction and functionality of the genetic modifications desired can be verified by any technique known by the person skilled in the art, notably:

-   -   selection thanks to the marker(s) present in the expression         cassette or in the vector;     -   targeting the genetic element introduced, for example by         sequencing, by PCR (“Polymerase Chain Reaction”) or         hybridization (“Southern blot” or “Northern blot”);     -   targeting the product of the gene sequence, for example by         immunological detection (“Western blot”) or by measuring the         associated activity, enzymatic activity for example.

As previously stated, the important elements to control gene expression are the promoters, placed upstream of the coding sequence whose expression is governed by the promoter. The genes can be thus expressed using inducible or constitutive promoters of variable force. According to a particular embodiment, the promoters used in the invention are constitutive promoters. These promoters can be homologous or heterologous. Within the context of the invention, the promoters commonly used by the person skilled in the art are, for example:

-   -   the promoter of gene TDH3 of S. cerevisiae, for example that         having sequence SEQ ID NO: 1;     -   the promoter of gene PFK2 of S. cerevisiae, for example that         having sequence SEQ ID NO: 4;     -   the promoter of gene PGI1 of S. cerevisiae, for example that         having sequence SEQ ID NO: 7;     -   the promoter of gene PMA1 of S. cerevisiae, for example that         having sequence SEQ ID NO: 10;     -   the promoter of gene PYK1 of S. cerevisiae, for example that         having sequence SEQ ID NO: 13;     -   the promoter of gene TEF1 of Ashbya gossypii, for example that         having sequence SEQ ID NO: 15 or S. cerevisiae

Other important elements to control gene expression are termination sequences, also called terminators, placed downstream of the coding sequence to be expressed. There again, they can be homologous terminators from the microorganism in question, or heterologous terminators, that is, artificial sequences or terminators from a source other than the host microorganism. Numerous termination sequences are available to the person skilled in the art, for example:

-   -   the terminator of gene CYC1 of Saccharomyces cerevisiae, for         example that having sequence SEQ ID NO: 3;     -   the terminator of gene PFK2 of S. cerevisiae, for example that         having sequence SEQ ID NO: 6;     -   the terminator of gene PGI1 of S. cerevisiae, for example that         having sequence SEQ ID NO: 9;     -   the terminator of gene ZWF1 of S. cerevisiae, for example that         having sequence SEQ ID NO: 12;     -   the terminator of gene PYK1 of S. cerevisiae, for example that         having sequence SEQ ID NO: 14;     -   the terminator of gene TEF1 of Ashbya gossypii, for example that         having sequence SEQ ID NO: 17.

In the context of the invention, the term “fungal microorganism” advantageously designates a yeast or a mushroom.

In the context of the invention, the term “fungal microorganism” means a “strain of fungal microorganism”. Indeed, advantageously, the genetically modified fungal microorganism, object of the present invention, is obtained from an isolated strain and at least partially characterized.

Illustratively and in reference to yeasts, “yeast” is understood to be a commercial product obtained by implementing a method for producing a yeast strain. Thus, yeasts having different properties can be obtained from a single strain, where these differences are connected with the production method implemented.

More precisely, the invention targets a yeast strain, in other words, a strain belonging to the phyla ascomycetes or basidiomycetes. Advantageously, the strain belongs to the genus Saccharomycetales, even more advantageously to the families Debaryomycetaceae, Dipodascaceae, or Saccaromycetaceae. According to a preferred embodiment, the strain belongs to the genus Yarrowia, Debaryomyces, Arsula, Scheffersomyces, Geotrichum, Pichia or Saccharomyces. For example, it can be of the species Yarrowia lipolytica, Debaryomyces hansenii or Saccharomyces cerevisiae.

According to a particular embodiment, the strain used for constructing a strain according to the invention or for constructing a process according to the invention is strain called “industrial”, as opposed to a strain called “laboratory”. Industrial yeast strains are those producible using industrial substrates as a carbon source. As an example, said carbon source can be molasses from sugar cane or sugar beet.

According to an advantageous embodiment, the microorganism is chosen with at least one of the following characteristics:

-   -   a capacity to at least partially produce frambinone from         tryosine; and/or     -   a limited capacity or no capacity to break tyrosine down into         tyrosol, p-hydroxyphenylacetaldehyde and/or         p-hydroxyphenylacetate.

Regarding the first characteristic, the microorganism to be genetically modified can have at least one endogenous enzymatic activity involved in the conversion of tyrosine into frambinone. According to a particular embodiment, 4-hydroxybenzalacetone can be converted into frambinone and naturally present a BAR activity, as detailed below:

The microorganism can be chosen for other characteristics of interest, notably for its biotechnological application (knowledge of the genome, tools available for genetic manipulation, etc.), its metabolism (notably respiratory and lipid favoring production of malonyl-CoA or acetyl-CoA, co-substrates for synthesis of frambinone), or conditions for its industrial use (aerobic growth, tolerance to stress and toxic compounds, etc.)

According to a particular embodiment, a microorganism of interest is chosen from the following list: Beauveria bassiana, Candida boidinii, Galactomyces candidum (Geotrichum candidum), Kloeckera saturnus, Kodamaea ohmeri (Pichia ohmeri), Komagataella pastoris (Pichia pastoris), Mucor nederlandicus (Mucor subtilissimus), Pichia membranifaciens, Schwanniomyces etchellsii (Pichia etchellsii), Torulaspora delbrueckii (Saccharomyces fermentati), Wickerhamomyces anomalus (Hansenula anomala), Yarrowia lipolytica (Candida lipolytica), Saccharomyces cerevisiae, Debaryomyces hansenii. Advantageously, it is Saccharomyces cerevisiae, Yarrowia lipolytica or Debaryomyces hansenii.

Fungal microorganisms of particular interest are Yarrowia lipolytica or Debaryomyces hansenii.

A microorganism according to the invention intended to produce frambinone using tryosine. Advantageously, such a microorganism has an improved capacity to produce frambinone from tyrosine, notably compared with strains already described or compared with the same microorganism that has not been genetically modified.

Frambinone is known to be produced from other substrates but tyrosine is clearly of interest from an economic point of view. In addition, it has been shown that adding tyrosine into a culture medium, advantageously a fermentation medium, was possible in terms of solubility and showed no noteworthy toxicity. In an appropriate manner and as seen in more detail in relation to the process according to the invention, the production of frambinone is done from tyrosine exogenous to the microorganism according to the invention, advantageously by addition into its culture medium. Typically, the concentration of tyrosine in the culture medium is greater than or equal to 50 mg/L, even greater than or equal to 100, 150, 200, 250, 300, 350, 400 or even 450 mg/L. In addition, it is advantageously less than or equal to 1 g/L, or even less than or equal to 950, 900, 850, 800, 750, 700, 650, 600, 550, 500 or even 450 mg/L.

According to another aspect, the invention also concerns the use of a microorganism as defined within the scope of the present application for the production of frambinone from tyrosine. Within the scope of the invention, production levels of frambinone by a microorganism never yet achieved are intended, advantageously a concentration in the culture medium of the microorganism greater than 4 mg/L, more advantageously greater than or equal to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even 20, 25 or 30 mg/L. In these concentrations and under the conditions of the process according to the invention, frambinone remains soluble with no toxicity vis-a-vis the microorganisms according to the invention.

According to a first characteristic, a microorganism according to the invention or implemented in a process according to the invention is genetically modified to have the capacity to produce or synthesize frambinone from tyrosine, or even an improved capacity to produce or synthesize frambinone from tyrosine.

According to the invention, the microorganism can naturally have the capacity to synthesize frambinone from tyrosine and the purpose of the genetic modifications is to improve this capacity. Alternatively, the organism does not naturally have the capacity to synthesize frambinone from tyrosine, and the purpose of the genetic changes is to confer this capacity.

As stated and shown in FIG. 1, a path for synthesis of frambinone from tyrosine involves 4 enzyme activities, namely:

-   -   a tyrosine ammonia lyase activity (EC 4.3.1.23), denoted TAL,         capable of deaminating tyrosine to form coumaric acid;     -   a 4-coumarate:CoA ligase activity (EC 6.2.1.12), denoted 4CL,         capable of catalyzing the grafting of a coenzyme A molecule         (CoA) on coumaric acid to form coumaroyl-CoA;     -   a benzalacetone synthase (EC 2.3.1.212), denoted BAS, capable of         converting coumaroyl-CoA into 4-hydroxybenzalacetone in the         presence of malonyl-CoA as co-substrate;     -   a benzalacetone reductase activity (EC 1.3.1.x), denoted BAR, to         reduce the 4-hydroxybenzalacetone to frambinone.

According to an advantageous embodiment, the microorganism according to the invention is genetically modified to ensure or improve the production of frambinone from tyrosol. Advantageously the genetic changes permit at least one of the four aforementioned enzymatic activities to increase. For example:

-   -   mutations in the genetic coding portion making it possible to         obtain an enzyme having the activity sought or with improved         activity, notably in terms of specificity and affinity with the         substrate;     -   modification of the regulatory sequences to increase the level         of expression of an endogenous gene encoding an enzyme with the         activity sought;     -   contribution of supernumerary copy(ies) of an endogenous gene         encoding an enzyme with the activity sought, optionally mutated         to improve activity;     -   contribution of one or more copies of at least one heterologous         gene (artificial or from another organism source) encoding an         enzyme with the activity sought, placed under control of         regulatory sequences adapted to produce the aforesaid enzyme.

Concerning TAL, the enzymes of the family of lyases having a predominant activity for tyrosine (tyrosine ammonia-lyase, EC 4.3.1.23) are rare. It often shows at least equal or even greater affinity for the substrate phenylalanine (phenylalanine/tyrosine ammonia-lyase, PAL/TAL, EC 4.3.1.25).

In certain cases, it may be desirable to choose a TAL enzyme with no or very little PAL activity so as to avoid the accumulation of the cinnamic acid intermediate, which can present a certain toxicity. Where the TAL enzyme chosen has well-known PAL activity, it may be advantageous to ensure that the microorganism additionally has C4H and/or CPR activity, and possibly to modify it to introduce sequences encoding the enzymatic activities useful in converting cinnamic acid to coumaric acid. Alternatively, one might mutate the TAL coding sequence to decrease the possibly associated PAL activity. In addition, an accumulation of cinnamic acid can exert negative feedback on TAL possibly necessitating a balanced expression of 4CL.

Note that enzymatic tests to evaluate TAL and PAL activities are well known to the person skilled in the art and are described in documents by Berner et al. (J. Microbiol., 2006, 188, 2666-73), Kyndt et al. (FEBS Lett., 2002 512, 240-44) and Rosier et al. (Plant Physiol., 1997, 113, 175-79).

Genes of interest that can be implemented within the scope of the invention are listed in Table 1 below:

Name of the Name Organism gene/Uniprot References RgTAL Rhodotorula PAL Vannelli et al., 2007, Metab. glutinis/ P11544 Eng. 9, 142-51. Rhodospor- SEQ ID US 6,521,748 idium NO: 18 Santos et al., 2011, Metab. Eng. toruloides 13, 392-400 Wu et al., 2013, J. Biotechnol. 167, 404-11 Wu et al., 2014, PLoS ONE 9, E101492 Jiang et al., 2005, Appl. Environ. Microbiol. 71, 2962-69 RsTAL Rhodobacter hutH Watts et al., 2004, sphaeroides Q3IWB0 Chembiochem Eur. J. Chem. Biol. 5, 500-507 Watts et al., 2006, Chem. Biol. 13, 1317-26 Louie et al., 2006, Chem. Biol. 13, 1327-1338 Schroeder et al., 2008, Phytochem. 69, 1496-1506 Xue et al., 2007, J. Ind. Microbiol. Biotechnol. 34, 599-604 Wang and Yu, 2012, J. Biotechnol. 157, 258-260 Wang et al., 2011, Metab. Eng. 13, 455-463 Zhang et al., 2006, J. Am. Chem. Soc. 128, 13030-31 RcTAL Rhodobacter Xue et al., 2007, J. Ind. capsulatus Microbiol. Biotechnol. 34, 599-604 Kyndt et al., 2002, FEBS Lett. 512, 240-44 SeTAL Saccharothrix sam8 Berner et al., 2006, J. espanaensis Q2EYY5 Bacteriol. 188, 2666-73. Choi et al., 2011, J. Ind. Microbiol. Biotechnol. 38, 1657-65 Kang et al., 2012, Microb Cell Fact 11, 153 CpTAL Chitinophaga Cpin_1853/ Schöner et al., 2014, Microb. pinensis FlxA Biotechnol. 7, 232-41 C7PAX7 TcTAL Trichosporon Vannelli et al., 2007, Enzyme cutaneum Microb. Technol. 41, 413-22

According to a particular embodiment, the TAL protein encoded by the gene introduced in the microorganism according to the invention has the sequence SEQ ID NO: 18, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or sequence identity SEQ ID NO: 18 and possessing TAL activity.

According to another particular embodiment, the TAL protein coding sequence introduced into the microorganism according to the invention has the sequence SEQ ID NO: 5, or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% sequence identity SEQ ID NO: 5. Its expression may be placed under the control of regulatory sequences SEQ ID NO.: 4 or SEQ ID NO: 6.

Concerning 4CL, it is a priori an enzyme that is not present in the fungal microorganisms considered in the present invention. Thus and advantageously, at least one copy of at least one heterologous gene is introduced.

Note that enzymatic tests to evaluate 4CL activities are well known to the person skilled in the art and are described in documents by Ehlting et al. (Plant J., 1999, 19, 9-20), Knobloch and Hahlbrock (Arch. Biochem. Biophys., 1977, 184, 237-48) and Lee and Douglas (Plant Physiol., 1996, 112, 193-205).

The majority of the 4-coumarate:CoA ligases (4CL; EC6.2.1.12) are found in plants. Genes of interest that can be implemented within the invention are listed in Table 2 below:

Name of the Name Organism gene/Uniprot References At4CL-1 Arabidopsis 4CL1 Ehlting et al., 1999, thaliana Q42524 (NCBI: Plant J. 19, 9-20 AAA82888.1) Hamberger and SEQ ID NO: 19 Hahlbrock, 2004, Proc. Natl. Acad. Sci. U.S.A. 101, 2209-14 Watts et al., 2004, Chembiochem Eur. J. Chem. Biol. 5, 500-507 Watts et al., 2006, BMC Biotechnol. 6, 22 Koopman et al., 2012, Microb Cell Fact 11, 155 Sydor et al., 2010, Appl. Environ. Microbiol. 76, 3361-63 Nt4CL-2 Nicotiana 4CL2 Beekwilder et al., 2007, tabacum O24146 (NCBI: Biotechnol. J. 2, 1270-79. U50846) Beekwilder et al., 2006, Appl. Environ. Microbiol. 72, 5670-72 Pc4CL-2 Petroselinum 4CL2 Leonard et al., 2008, Mol. crispum P14913 Pharm. 5, 257-65 (GenBank: Lim et al., 2011, Appl. CAA31697.1) Environ. Microbiol. 77, 3451-60 Wu et al., 2014, PLoS ONE 9, E101492 Xu et al., 2011, Metab. Eng. 13, 578-87. Leonard et al., 2005, Appl. Environ. Microbiol. 71, 8241-48. Yan et al., 2005, Appl. Environ. Microbiol. 71, 5610-13 Lithospermum Q42880 Yazaki et al., 1997, erythrorhizon Biosci. Biotechnol. Biochem. 61, 1995-2003 Populus trichocarpa × P. deltoides Glycine max ScCCL Streptomyces SCO4383 Kaneko et al., 2003, J. coelicolor Q9K3W1 Bacteriol. 185, 20-27 Miyahisa et al., 2005, Appl. Microbiol. Biotechnol. 68, 498-504 Cp4CL Chitinophaga Cpin_1877/FlxY Schöner et al., 2014, pinensis C7PB01 Microb. Biotechnol. 7, 232-41

According to a particular embodiment, the 4CL protein encoded by the gene introduced in the microorganism according to the invention has the sequence SEQ ID NO: 19, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or identity with sequence SEQ ID NO: 19 and possessing 4CL activity.

According to another particular embodiment, the 4CL protein coding sequence introduced into the microorganism according to the invention has the sequence SEQ ID NO: 8, or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% sequence identity SEQ ID NO: 8. Its expression may be placed under the control of regulatory sequences SEQ ID NO.: 7 and/or SEQ ID NO: 9.

It should be noted that the accumulation of coumaryl-CoA generated under 4CL action can engender the undesirable formation of phloretic acid and inhibit TAL activity. It is therefore important that the microorganism according to the invention has appropriate BAS activity (catalyzing the next step in synthesizing frambinone).

Concerning BAS, it is a priori an enzyme that is not present in the fungal microorganisms considered in the present invention. Thus and advantageously, at least one copy of at least one heterologous gene is introduced.

Note that enzymatic tests to evaluate BAS activities are well known to the person skilled in the art and are described in documents by Abe et al. (Eur. J. Biochem., 2001, 268, 3354-59 and Morita et al., 2010). Morita et al. (Acad. Sci. 2010, 107, 669-673).

Benzalacetone synthases (BAS; EC2.3.1.212) are part of the PKS (PolyKetone synthase) family that also includes chalcone synthases (CHS; involved for example in the synthesis of naringenin) and stilbene synthases (STS; involved for example in the synthesis of resveratrol). These enzymes accept coumaroyl-CoA and other substrates and catalyze a condensation with malonyl-CoA. Malonyl-CoA is an intermediate in the synthesis of fatty acids and its formation requires ATP. While CHS and STS add three malonyl-CoA units, BAS adds only one. However, the BAS enzymes described also have CHS activity. Genes of interest that can be implemented within the invention are listed in Table 3 below:

Name of the Name Organism gene/Uniprot References RpBAS Rheum BAS SA Abe et al., 2001, Eur. J. palmatum Q94FV7 Biochem. 268, 3354-59. SEQ ID NO: 20 Shimokawa et al., 2012, Front. Plant Sci. 3. Abe et al., 2003, J. Biol. Chem. 278, 25218-26. Abe et al., 2007, Bioorg. Med. Chem. Lett. 17, 3161-66. Jez et al., 2001, J. Ind. Microbiol. Biotechnol. 27, 393-398. Morita et al., 2010, Proc. Natl. Acad. Sci. 107, 669-673. RiPKS4 Rubus idaeus PKS4 Kumar and Ellis, 2003, B0LDU5 Phytochemistry 62, 513-26. Zheng and Hrazdina, 2008, Arch. Biochem. Biophys. 470, 139-145. Zheng et al., 2001, Plant Mol. Biol. 46, 1-15 Beekwilder et al., 2007, Biotechnol. J. 2, 1270-79. PKS1 Wachendorfia Brand et al., 2006, Planta thyrsiflora 224, 413-28. PKS1 Polygonum My et al., 2009, Planta 229, cuspidatum 1077-86 PKS2 Polygonum Ma et al., 2009, Planta 229, cuspidatum 457-69.

According to a particular embodiment, the protein BAS encoded by the gene introduced into the microorganism according to the invention has the sequence SEQ ID NO: 20, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or identity with sequence SEQ ID NO: 20 and possessing BAS activity.

According to another particular embodiment, the BAS protein coding sequence introduced into the microorganism according to the invention has the sequence SEQ ID NO: 11, or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% identity with sequence SEQ ID NO: 11. Its expression may be placed under the control of regulatory sequences, particularly a sequence promoter for SEQ ID NO.: 10 or SEQ ID NO: 13 and/or sequence terminator SEQ ID NO: 12 or SEQ ID NO: 14.

According to another particular embodiment, at least two BAS coding sequences, for example two copies of the same sequence, are introduced into the microorganism according to the invention. It should be noted that they can be placed under the control of various regulatory sequences.

The last step in synthesizing frambinone is the reduction of the α,β-double bond in p-hydroxybenzalacetone, which requires NADPH, catalyzed by benzalacetone reductase (BAR; EC 1.3.1.x). Only two enzymes with this activity have been identified to date. However, certain microorganisms have been reported to have endogenous BAR activity, E. coli and S. cerevisiae for example (Beekwilder et al., 2007, Biotechnol. J. 2, 1270-79). The first enzyme is described in document GB 2 416 769: a protein of 309 amino acids was isolated from raspberry protein fractions with BAR activity. It is homologous with isoflavone reductases (EC 1.3.1.45) and is capable of converting p-hydroxybenzalacetone into frambinone in in vitro tests carried out with purified enzyme. In 2011, Koeduka et al. (Biochem. Biophys. Res. Common. 412, 104-108) have identified a ketone/zingerone synthase of R. idaeus with BAR activity (RiRZS1, Uniprot G1FCG0). The purified protein effectively converts p-hydroxybenzalacetone into frambinone in an enzymatic test.

Note that enzymatic tests to evaluate BAR activity are well known to the person skilled in the art and are described in Koeduka et al. document (Biochem. Biophys. Res. Commun., 2011, 412, 104-108).

According to a particular embodiment, the protein BAR is encoded by a gene introduced into the microorganism according to the invention and with the sequence SEQ ID NO: 21, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or sequence identity SEQ ID NO: 21 and possessing BAR activity.

According to another particular embodiment, the BAR protein coding sequence is introduced into the microorganism according to the invention has the sequence SEQ ID NO: 2, or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% identity with sequence SEQ ID NO: 2. Its expression may be placed under the control of regulatory sequences, particularly sequences SEQ ID NO.: 1 and/or SEQ ID NO: 3.

According to a particular embodiment, the fungal microorganism according to the invention is naturally devoid of at least one enzymatic activity among 4CL and BAS, or even the 2.

Advantageously, the fungal microorganism according to the invention includes at least one heterologous sequence encoding the enzyme 4-coumarate:CoA ligase (4CL) or benzalacetone synthase (BAS), advantageously enzymes 4CL and BAS.

According to a preferred embodiment, said sequence codes a 4CL enzyme having the sequence SEQ ID NO: 19, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or identity with sequence SEQ ID NO: 19 and possessing 4CL activity. Preferably, this sequence comprises the sequence SEQ ID NO: 8, or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% sequence identity SEQ ID NO: 8. It can also comprise the sequence SEQ ID NO: 7, advantageously located upstream of the 4CL coding sequence, and/or the sequence SEQ ID NO: 9, advantageously located downstream of the 4CL coding sequence.

According to another preferred embodiment, said sequence encodes a BAS enzyme showing the sequence SEQ ID NO: 20, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or identity with sequence SEQ ID NO: 20 and possessing BAS activity. Preferably, said sequence includes sequence SEQ ID NO: 11 or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% sequence identity SEQ ID NO: 11. It can also comprise the sequence SEQ ID NO: 10 or SEQ ID NO: 13, advantageously located upstream of the BAS coding sequence, and/or the sequence SEQ ID NO: 12 or SEQ ID NO: 14, advantageously located downstream of the BAS coding sequence.

According to a particular embodiment, the fungal microorganism according to the invention comprises at least two heterologous sequences encoding the enzyme benzalacetone synthase (BAS), advantageously from the same source, even more advantageously of the same coding sequence but possibly placed under the control of different regulatory sequences.

According to another embodiment, the fungal microorganisms within the present invention do not present at least one of the following activities: TAL, 4CL, BAS and/or BAR.

Advantageously, the fungal microorganism according to the invention comprises at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia lyase (TAL) or benzalacetone reductase (BAR), advantageously enzymes TAL and BAR.

According to a preferred embodiment, said sequence encodes a TAL enzyme with the sequence SEQ ID NO: 18, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or sequence identity SEQ ID NO: 18 and possessing TAL activity. Preferably, this sequence comprises the sequence SEQ ID NO: 5, or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% sequence identity SEQ ID NO: 5. It can also comprise the sequence SEQ ID NO: 4, advantageously located upstream of the TAL coding sequence, and/or the sequence SEQ ID NO: 6, advantageously located downstream of the TAL coding sequence.

According to a preferred embodiment, said sequence encodes a BAR enzyme with the sequence SEQ ID NO: 21, or a protein sequence with at least 70%, or even 80, 85, 90, 95 or even 99% homology or sequence identity SEQ ID NO: 21 and possessing BAR activity. Preferably, this sequence comprises the sequence SEQ ID NO: 2, or a sequence with at least 60%, even 70, 80, 85, 90, 95 or even 99% identity with sequence SEQ ID NO: 2. It can also comprise the sequence SEQ ID NO: 1, advantageously located upstream of the BAR coding sequence, and/or the sequence SEQ ID NO: 3, advantageously located downstream of the BAR coding sequence.

According to a particular embodiment, the fungal microorganisms referred to by the present invention include at least one heterologous sequence encoding the enzymes 4-coumarate:CoA ligase (4CL) and benzalacetone synthase (BAS), and at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia-lyase (TAL) and benzalacetone reductase (BAR).

A particular strain of Saccharomyces cerevisiae with these characteristics, and thus a path to converting tyrosine into functional frambinone and appropriate for the target applications, is the industrial strain RK4, registered with the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) dated Jun. 1, 2016 under number I-5101. This was obtained by chromosomal integration, at the level of HO locus of the strain registered with the CNCM dated Sep. 4, 2008 under number I-4071, expression cassettes encoding these 4 enzymes, as described below (See examples).

Another particular strain of Saccharomyces cerevisiae with these same characteristics is the industrial strain RK5, registered with the CNCM on Apr. 26, 2017 under number I-5199. This was obtained from strain RK4, by excising the kanMX 4 gene into the expression cassette (see examples below).

With respect to the path of synthesizing frambinone, the fungal microorganisms according to the invention may undergo other genetic modifications, such as for example:

-   -   any means to establish or increase the capacity of the organism         to synthesize the frambinone from phenylalanine, for example,         via the introduction of a gene encoding a PAL enzyme. As stated,         PAL enzymes are related to the TAL enzymes described above.         Optionally, a TAL enzyme also showing PAL activity may be used.     -   any means to establish or increase the capacity of the organism         to convert cinnamic acid to acid coumaric, for example via the         introduction of a gene encoding a C4H enzyme, for example,         optionally in combination with a gene encoding a CPR enzyme.     -   any means to improve the capacity of the organism to produce         malonyl-CoA, for example as reported in Y. lipolytica (Qiao et         al., 2015, Metab. Eng. 29: 56-65) or to overproduce Acetyl-Cohas         carboxylase ACC1 in S. cerevisiae (Shin et al., 2012, Enzyme         Microb. Technol. 51, 211-216).

Thus and according to a particular embodiment, the microorganism used according to the invention comprises at least one heterologous or supernumerary sequence encoding the enzyme phenylalanine ammonia lyase (TAL) or 4-(C4H), advantageously TAL and C4H enzymes.

According to a second advantageous characteristic, one target fungal microorganism of the invention is earmarked in its path to break down tyrosine as illustrated in FIG. 2. According to this path, called “path of breaking tyrosine down into tyrosol”, tyrosine is converted to p-hydroxyphenylacetaldehyde, which can continue in that state or be converted either into tyrosol or into p-hydroxyphenylacetate. Thus and advantageously, a target fungal microorganism within the invention has a limited capacity or no capacity to break down or to convert tyrosine into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate.

It is evident, in the scope of the present application, that the predominant path to breaking down tyrosine into the fungal microorganisms of interest was the path for breaking tyrosine down into tyrosol, as shown in FIG. 2.

Said capacity can be evaluated as described in the section “Examples of Implementation” below, cultivating of fungal microorganisms potentially of interest in the production of frambinone, genetically modified or not, in the presence of tyrosine and following the production of tyrosol, for example by HPLC. Typically, a microorganism may prove to be of interest if in the conditions described in the experimental part, namely in conditions of aerobic fermentation conducted in the medium, advantageously an inorganic medium, for example composed of 1.7 g/L of YNB (Difco™), 5 g/L of ammonium sulfate, 2.7 g/L potassium phosphate and 20 g/L of dextrose, and containing tyrosine, advantageously at a level of 300 mg/L, the quantity of tyrosol produced is less than or equal to 150 mg/L, advantageously less than or equal to 100 mg/L, even 50, 40, 30, 20 mg/L or even 10 mg/L.

Alternatively and as is clear from FIG. 2, strains of the fungal microorganisms of interest can be selected on the basis of their hydroxyphenyl pyruvate decarboxylase (HPPDC) activity, a method of measurement described in the examples of implementation, below.

Advantageously, the HPPDC activity in the microorganism according to the invention is less than or equal to 2×10⁻⁶ KAT per g of protein, advantageously less than or equal to 1×10⁻⁶ KAT per g of protein, or even to 5×10⁻⁷ KAT per g of protein. In the scope of the invention, it refers to the proteins extracted from the microorganism.

More specifically, the HPPDC activity measured in a microorganism according to the invention is preferentially less than or equal to 10⁻⁵ KAT per g of protein, advantageously less than or equal to 5×10⁻⁶, 4×10⁻⁶, 3×10⁻⁶, 2×10⁻⁶, 1×10⁻⁶ KAT per g of protein, or even less than or equal to 9×10⁻⁷, 8×10⁻⁷, 7×10⁻⁷, 6×10⁻⁷, 5×10⁻⁷ KAT per g of protein. According to a particular embodiment, it is less than or equal to 4×10⁻⁷ KAT per g of protein, advantageously less than or equal to 3×10⁻⁷ KAT per g of protein, even more advantageously less than or equal to 2×10⁻⁷, 1.5×10⁻⁷, 1×10⁻⁷, or even 0.5×10⁻⁷ KAT per g of protein.

Thus and according to the invention, the microorganism is selected for its low or nil capacity to break down tyrosine, assessed according to one of the two methods mentioned above. As already said, this selection of adapted microorganisms can be performed before or after the genetic modification of the said microorganism.

According to a first embodiment, the microorganism is selected for its naturally limited capacity or incapacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate.

Alternatively, a microorganism of interest, in particular for its capacity to synthesize frambinone, is subjected to genetic modifications to reduce or even eliminate its capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate.

As shown in FIG. 2, this can be achieved by inhibiting or by inactivating one of the steps that ensures the transformation of tyrosine to tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate, particularly the step of deamination, decarboxylation, or even reduction.

As already stated, different means are available to the person skilled in the art to achieve this inhibition or inactivation by genetic modification of the microorganism, including the chromosomal insertion of genetic elements either exogenous at the level of the regulatory regions in such a way as to interfere with the expression of the target gene, either at the level of the coding sequence in a manner to prevent the production of the product of the gene or to lead to the production of a truncated and/or inactive protein. It is also possible to mutate the target gene at the level of critical sequences for its expression or its activity.

According to a particular embodiment, the target gene is inactivated using a cassette capable of expressing a marker (Goldstein and McCusker, 1999, Yeast Chichester Engl. 15, 1541-53; Güldener et al., 2002, Nucleic Acids Res. 30, e23; Güldener et al., 1996, Nucleic Acids Res. 24, 2519-24.; Janke et al., 2004, Yeast 21, 947-62.; Sauer, 1987, Mol. Cell. Biol. 7, 2087-96), at the ends of which the 5′ and 3′ regions of the target gene are inserted so as to allow the homologous recombination and replace the coding portion of the gene by the expression cassette of the marker.

According to an advantageous embodiment, the fungal microorganism according to the invention is deactivated at the level of the activity involved in the decarboxylation of the hydroxyphenyl pyruvate. In particular, and concerning S. cerevisiae, at least 3 genes have been described as being involved in this activity, i.e., ARO10, PDC5 and PDC6 (Hazelwood et al., 2008; Appl. Environ. Microbiol. 74, 2259-66; Kneen et al., 2011, FEBS J. 278, 1842-53; Vuralhan et al., 2005, Appl. Environ. Microbiol. 71, 3276-84.; Vuralhan et al., 2003, Appl. Environ. Microbiol. 69, 4534-41). Advantageously, at least one of the genes encoding phenylpyruvate decarboxylase Aro10, pyruvate decarboxylase PDC5 and pyruvate decarboxylase PDC6 is deactivated. According to a particular embodiment, genes encoding phenylpyruvate decarboxylase Aro10, pyruvate decarboxylase PDC5 and pyruvate decarboxylase PDC6 are deactivated.

Alternatively, the deaminase(s) involved in the first step of the path of breaking down tyrosine are deactivated. In relation to S. cerevisiae, this can be deaminase Aro8 and/or deaminase Aro9, or their equivalents in other fungal microorganisms.

Similarly, the alcohol dehydrogenase(s) (denoted ADH) involved in the third step of the path to break tyrosine down into tyrosol can be targeted.

Thus and according to a particular aspect, the present invention seeks a fungal microorganism comprising at least one mutation or deletion in at least one of the genes encoding the following enzymes: deaminase Aro8, deaminase Aro9, decarboxylase Aro10, decarboxylase PDC5, decarboxylase PDC6, alcohol dehydrogenase (ADH). In the scope of the invention, the aforesaid mutations and/or deletion entail a reduction or even the elimination of the capacity of the organism to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate. Advantageously, the aforesaid microorganism also has a capacity to synthesize frambinone from tyrosine, possibly through the introduction of genetic modifications as described above.

According to a particular embodiment, such a microorganism is a strain of Saccharomyces cerevisiae which has the capacity to produce frambinone and to inactivate the gene ARO10 encoding a decarboxylase. This microorganism therefore has a path for converting tyrosine into functional frambinone and a limited capacity or no capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate as described above.

For example, this could be the industrial strain RK8, filed with the CNCM on Apr. 26, 2017 under number I-5200. This was obtained from strain RK5, by inserting a cassette allowing inactivation of the ARO10 gene (see examples of implementation below).

According to another aspect, the present invention relates to the use of a fungal microorganism with the capacity to produce the frambinone from tyrosine and a limited capacity or no capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate, advantageously as described above and notably strain I-5200, for the production of frambinone, advantageously by aerobic fermentation.

In other words, the invention also relates to a process for the production of frambinone comprising the culture of a fungal microorganism with the capacity to produce frambinone from tyrosine and a limited capacity or no capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate, advantageously as described above, particularly strain I-5200, in a medium comprising tyrosine. According to a particular embodiment, tyrosine is added into the culture medium at a concentration of between 50 and 450 or 500 mg/L, for example on the order of 300 mg/L. Optionally, the culture medium can also be fortified with coumaric acid and/or phenylalanine.

Alternatively, the microorganism implemented within the invention naturally has a certain capacity to break tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate, but this path for breaking it down is inhibited by the addition of a repressor to this path, an inhibitor of one of the enzymes involved in this path for instance.

Thus and according to another aspect, the invention relates to a process for the production of frambinone comprising a fungal microorganism culture with the capacity to produce frambinone from tyrosine in a medium consisting of tyrosine and at least one repressor of the path for breaking tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate. Optionally, it is a microorganism genetically modified to confer or increase the capacity of the organism to produce the frambinone from tyrosine, as described above.

According to a particular embodiment, the microorganism implemented is the industrial strain Saccharomyces cerevisiae RK4, registered with the CNCM (National Collection of Microorganism Cultures, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on Jun. 1, 2016 under number I-5101.

Alternatively, the microorganism implemented is the industrial strain Saccharomyces cerevisae RK5, registered with the CNCM (National Collection of Microorganism Cultures, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on Apr. 26, 2017 under number I-5199.

According to a particular embodiment, the repressor of the path for breaking tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate is chosen from among glutamate, glutamine, or one of their derivatives, advantageously glutamate. According to a particular embodiment, said repressor, particularly glutamate, is added into the culture medium at a concentration greater than or equal to 0.5 g/L, even 1, 2 or 3 g/L, on the order of 2 g/L for example.

In the scope of the invention, the fungal microorganism is placed in a culture under conditions favoring the production of frambinone. Conditions particularly suitable for the culture are the following:

-   -   in aerobiosis.     -   in a growth medium suitable for fermentation, advantageously an         inorganic medium, for example, composed of 1.7 g/L of YNB         (Difco™), 5 g/L of ammonium sulfate, 2.7 g/L potassium phosphate         and 20 g/L of dextrose;     -   from several hours to several days;     -   with a pH between 5 and 7, equal to 6 for instance;     -   at a temperature between 25° C. and 32° C., equal to 30° C. for         instance.

As previously stated and under these conditions, production levels of frambinone were observed that had not previously been achieved, the culture medium showing a concentration of frambinone advantageously greater than 4 mg/L, more advantageously greater than or equal to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even 20, 25 or 30 mg/L.

In a manner known by the person skilled in the art, the frambinone thus produced can be isolated from the culture medium.

The invention will be described in greater detail in the following examples of implementation, supported by the attached figures. These examples of the invention are non-limiting and provided for illustrative purposes only.

Without more precision, it is deemed that the person skilled in the art, using descriptions and examples, will be able to implement and use the microorganisms according to the invention and the methods claimed.

LEGENDS FOR THE FIGURES

FIG. 1: Path for the biosynthesis of frambinone

(1) L-tyrosine (2) p-coumaric acid (3) coumaroyl-CoA (4) p-hydroxybenzalacetone (5) frambinone (6) malonyl-CoA (7) phenylalanine (8) cinnamic acid

TAL: tyrosine ammonia-lyase, 4CL: 4-coumarate-CoA ligase, BAS: benzalacetone synthase, BAR: benzalacetone reductase, PAL: phenylalanine ammonia-lyase, C4H: cinnamate 4 hydroxylase

FIG. 2: Path for the biosynthesis of tyrosine

This consists of the deamination (Aro8/9) and decarboxylation (Aro10/PDC5/PDC6) steps leading to the formation of p-hydroxyphenylacetaldehyde. The latter may be reduced using an alcohol dehydrogenase (ADH) into tyrosol or oxidized to p-hydroxyphenylacetate. Possible inactivation of the path is indicated by a cross.

FIG. 3: Diagram of the HO locus modified in the genome of strains S. cerevisiae RK4, RK5 and RK8 and the cassette allowing modification of locus ARO10

-   -   A) Diagram of integrated cassettes at HO locus allowing the         synthesis of frambinone. Five gene expression cassettes and a         marker cassette have been integrated at the level of HO locus by         using different regions of overlapping recombination (RR1-5)         between the cassettes for the in vivo assembly. In strains RK5         and RK8 the cassette marker (pTEF-KanMX-tTEF) is absent.     -   B) Diagram of the cassette allowing inactivation of gene ARO10         and conferring resistance to hygromycin.

FIG. 4: Production of frambinone by strain S. cerevisiae RK4 after 7 days

The concentration of frambinone (mg/L) was determined after 7 days of culture as a function of the substrate (tyrosine synthesized by the cell from glucose and ammonium sulfate (called de novo process), tyrosine or coumaric acid added to the culture medium).

FIG. 5: Production of tyrosol by strain S. cerevisiae RK4 after 7 days

The concentration of frambinone (mg/L) was determined after 7 days of culture as a function of the substrate (tyrosine synthesized by the cell from glucose and ammonium sulfate (called de novo process), tyrosine or coumaric acid added to the culture medium).

FIG. 6: HDPPC activity in cellular extracts (strain S. cerevisiae RK4, RK5 and RK8) after 16 hours of fermentation in the synthetic medium containing tyrosine with or without glutamate

The hydroxy-phenylpyruvate (HDPPC) activity (expressed in nmol/min/mg protein) was determined in the cell extract after 16 hours of fermentation in a synthetic medium containing 0.3 g/L of tyrosine and optionally 2 g/L of glutamate.

EXAMPLE EMBODIMENTS

The present invention will be further illustrated with respect to a strain of Saccharomyces cerevisiae genetically modified to express 4 heterologous genes encoding enzymes TAL, 4CL, BAS and BAR integrated at its HO locus and effectively producing frambinone from tyrosine in a medium enriched by glutamate, or with a derived strain showing an inactivated ARO10 gene. These examples are in no way limiting.

I) MATERIAL AND METHODS

Generation of Expression Cassettes and Recombinant Strains

To synthesize frambinone from tyrosine, it was chosen to express four heterologous genes in Saccharomyces cerevisiae as shown in the Table 4 below:

TABLE 4 Heterologous genes used to establish a path for synthesizing frambinone with S. cerevisiae Sequence (codon-optimized for Enzyme Source Reference expression with S. cerevisiae) TAL Rhodotorula Uniprot SEQ ID NO: 5 glutinis P11544 4CL Arabidopsis Uniprot SEQ ID NO: 8 thaliana Q42524 BAS SA Rheum Uniprot SEQ ID NO: 11 palmatum Q94FV7 BAR Rubus idaeus Uniprot SEQ ID NO: 2 G1FCG0

The encoding sequences were “codon-optimized” with S. cerevisiae. The corresponding sequences are shown in Table 4 above.

They were cloned between the proponents and terminators to ensure their expression. Five gene expression cassettes were built (Table 5 below) and, in addition to a cassette marker, were integrated into the genome of industrial strain S. cerevisiae filed with the CNCM Sep. 4, 2008 under number I-4071, at the HO locus using the modular cassette integration technique (FIG. 3A). The resulting strain, RK4, was registered with the CNCM (Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) on Jun. 1, 2016, under number I-5101.

TABLE 5 Gene expression cassettes and marker used in strain RK4 position of relative SEQ Coding SEQ SEQ cassette integration Promoter* ID NO: sequence ID NO: Terminator* ID NO: BAR 1 TDH3 1 RiRZS1 2 CYC1 3 TAL 2 PFK2 4 RgTAL 5 PFK2 6 4CL 3 PGI1 7 At4CL-1 8 PGI1 9 BAS1 4 PMA1 10 RpBAS 11 ZWF1 12 BAS2 5 PYK1 13 RpBAS 11 PYK1 14 marker 6 TEF1 15 kanMX 16 TEF1 17 *S. cerevisiae with the exception of TEF1 (Ashbya gossypii) Ri = Rubus idaeus Rg = Rhodotorula glutinis At = Arabidopsis thaliana Rp = Rheum palmatum

From the RK4 strain described above, the RK5 strain was obtained by elimination of the kanMX cassette marker. This was done by expressing the Cre recombinase that leads to excising the kanMX marker which is flanked by loxP sites (Steensma and Linde, 2001, Yeast, 18(5): 469-72). This is the strain registered with the CNCM on Apr. 26, 2017 under number I-5199.

An inactivation cassette of the gene ARO10 was then constructed (FIG. 3B). It is composed of a cassette marker in hygromycin, pTEF1short-HPH-tTEF1 (pTEF1 short: SEQ ID NO: 22; hph: SEQ ID NO: 23; tTEF1: SEQ ID NO: 24), flanked by sequences homologous to the promoter (pARO10: SEQ ID NO: 25) and the terminator (tARO10; SEQ ID NO: 26) of ARO10, both upstream and downstream, respectively. A RNA sequence guide specific to the ARO10 gene has been cloned into a plasmid to express Case9p and said RNA in S. cerevisiae. The RK5 strain described above has been co-transformed with this plasmid and the inactivation cassette of gene ARO10. Positive clones (resistant to hygromycin) have been selected and verified for the inactivation of all alleles of ARO10.

The resulting strain RK8 was registered with the CNCM on Apr. 26, 2017 under number I-5200.

HPLC Measurements of the Path's Intermediates and Products

Frambinone, tyrosol and other intermediates of the path were analyzed and quantified by two HPLC methods called “long method” and “ACE_29,” respectively. HPLC devices and their parameters are summarized in Table 6 below and allow the separation of the frambinone and tyrosol from other compounds. For quantification, calibration was done with standard solutions between 0.1 and 300 mg/L. The samples of the yeast cultures were centrifuged (>15,000×g, 10 min), and the supernatant filtered through a 0.45 μm filter before injection into the HPLC.

TABLE 6 HPLC devices and parameters Long method ACE_29 HPLC system Shimadzu LC20AD, PDA detector Column Polar Advantage II C18-PFP [MK] 3 × 150 mm 3 μm (reverse phase) Eluent A Water + 0.1% formic acid Eluent B Methanol Elution Isocratic: Gradient 82% eluent A and 18% eluent B Temperature 30° C. of the column Flow Rate 0.4 mL/min 0.35 mL/min Injection volume 20 μL; Temperature of the 10° C. automatic sampler PDA detection 200-300 nm 200-400 nm Quantification Quantification at λ 280 nm at λ 280 nm Enzymatic Detection of Hydroxyphenyl Pyruvate Decarboxylase (HPPDC) Activity

To quantify the enzymatic activity of the decarboxylation of hydroxyphenyl pyruvate (HPPDC activity), an enzymatic test paired with a crude cell extract has been developed. The cell extract was prepared from an overnight culture (16 h) of the strain of interest in a synthetic medium composed of 1.7 g/L of YNB (Difco™), 5 g/L of ammonium sulfate, 2.7 g/L of potassium phosphate and 20 g/L of dextrose. The environment was also supplemented by 300 mg/L L-tyrosine and optionally different nitrogen sources (for example, L-glutamate). After aerobic growth at 30° C., the cells were harvested by centrifugation (5000×g, 4 min, 4° C.) and washed twice in a wash buffer (10 mM phosphate potassium, 2 mM EDTA, pH 6.8). The packed cell was taken up in an extraction buffer (100 mM potassium phosphate, 2 mM magnesium chloride, 1 mM DTT, 1× cOmplete™ proteinase inhibitors, pH 6.8) and the cells were broken with a “FastPrep” disruptor (with 0.45 mm glass beads; four 30 s to 6 m/s cycles, and 1 min on ice). The cell debris was removed by centrifugation and the supernatant used as crude cell extract. The protein concentration was determined using the “Uptima BC Assay Protein Quantification Kit” according to the manufacturer's instructions.

The enzyme assay was carried out as described by Kneen et al. (2011, FEBS J. 278, 1842-53), with minor modifications. The assay couples the HPPDC reaction (decarboxylation of hydroxyphenyl pyruvate (HPP) in hydroxyphenylacetaldehyde) with a second reaction (oxidation of hydroxyphenylacetaldehyde in hydroxyphenyl ethanol/tyrosol) catalyzed by the auxiliary enzyme alcohol dehydrogenase (ADH). The ADH activity leads to reduction of NADH to NAD⁺ which can be followed thanks to the decrease of absorption at 340 nm in a spectrophotometer. The reaction mixture (1 mL) contained 100 mM potassium phosphate, 1 mM magnesium chloride, 0.5 mM thiamine pyrophosphate, 0.1 mM of NADH, 0.5 U of ADH with horse liver, 4 mM of HPP and the crude cell extract equivalent to approximately 200 μg of total protein. Reactions were measured at 32° C. and pH 6.8. The reaction was initiated by the addition of the substrate HPP.

II/ RESULTS

1/ Production of Frambinone and Other Metabolites from Tyrosine or Coumaric Acid as Substrate by Strain RK4.

Fermentation tests were conducted with strain RK4, cultivated in an inorganic medium composed of 1.7 g/L of YNB (Difco™), 5 g/L of ammonium sulfate, 2.7 g/L of potassium phosphate and 20 g/L of dextrose. Optionally, the medium can contain 300 mg/L of tyrosine or 100 mg/L of coumaric acid. As suggested by Ayuso et al. (2016, Microb. Cell Factories 15. doi:10.1186/s12934-016-0446-2), fermentation tests were conducted under aerobic conditions to optimize the production of frambinone.

As shown in FIG. 4, strain RK4 can synthesize approximately 6 mg/L of frambinone from tyrosine. When coumaric acid is used as substrate, the concentration of frambinone in mediums reaches approximately 14 mg/L. These results are in agreement with those of the prior art and confirm the fact that the production of frambinone from aromatic amino acids is less efficient than from coumaric acid. However, in view of the price of the substrates used for bioconversion, industrial applications are only profitable when tyrosine is used rather than coumaric acid.

A last point of these results concerns the de novo synthesis of frambinone by the constructed strain RK4. Note that the frambinone concentration is approximately the same that observed in the presence of tyrosine. It has been hypothesized that this is probably linked to the regulation of the biosynthesis of tyrosine by extracellular tyrosine, and also to diverting this amino acid by a degradation pathway.

2/ Breaking Tyrosine Down into Tyrosol in Strain RK4

Breaking tyrosine down into tyrosol is a well-known method (FIG. 2) (Hazelwood et al., 2008, Appl. Environ. Microbiol. 74, 2259-66). The first step is transamination of tyrosine resulting in the formation of hydroxyphenyl pyruvate. This compound is then decarboxylated into hydroxyphenylacetaldehyde (EC: 4.1.1.80). Finally, the aldehyde function is reduced to form a hydroxylated molecule called tyrosol (EC: 1.1.1.90).

The production of tyrosol during the fermentation of the strain RK4 was followed. FIG. 5 shows the concentrations of tyrosol in different mediums after 7 days of fermentation. Results confirm that the major part of the tyrosine provided is used for the production of tyrosol.

3/ Inhibition of Hydroxyphenyl Pyruvate Decarboxylase (HPPDC) Activity.

To reduce production of tyrosol in strain RK4, it was decided to inhibit the activity of the enzyme involved in the decarboxylation of hydroxyphenyl pyruvate by adding into the fermentation compounds that reduce HPPDC activity.

Glutamate was selected to be added to the medium to reduce the HPPDC activity. To be certain that adding this supplemental amino acid yields a reduction in enzyme activity, the enzymatic test described above was performed. FIG. 6 shows activity in the cell extraction of RK4 after culture in the fermentation medium containing tyrosine with or without 2 g/L of glutamate. Results confirm reduced HPPDC activity in cells fermenting in the presence of glutamate.

HPPDC activity of strain RK8 was measured at the same time. FIG. 6 states that the deletion of the ARO10 gene encoding for decarboxylase permits the reduction of HPPDC activity in the same proportions as strain RK4 in the presence of glutamate.

FIG. 6 also reveals that, as expected, strains RK4 and RK5 (from which strain RK8 derives) have the same level of HPPDC activity in the presence of tyrosine.

4/ Impact of Inhibition of Breaking Tyrosine Down on the Production of Frambinone and Tyrosol

In the first of a series of experiments, the production of tyrosol and frambinone by strain RK4 was followed by HPLC (“Long Method”) under both conditions (with or without glutamate). The concentrations of tyrosol observed demonstrate that the reduction of HPPDC activity also reduces the formation of tyrosol: a reduction of 27% in the tyrosol concentration was observed in response to glutamate, in line with the reduction of the HPPDC activity. At the same time, the production of frambinone increased by 40%. This data strongly suggests that the reduction of HPPDC activity reduces the production of tyrosol, thus rendering the tyrosine more available for the frambinone path.

In a second series of experiments, determination of the levels of frambinone (Table 7) and tyrosol (Table 8) produced by the three strains of S. cerevisiae built (RK4, RK5 and RK8) was assessed by HPLC according to the two methods described (“long method” and “ACE_29”). The ACE_29 method uses a new generation column with increased separation and compound resolution abilities, thus reducing the risk of co-elution, notably of frambinone, with other compounds as compared to the “long method”.

The results obtained with the “long method” from fermentation in a culture medium containing glutamate show an increase in the frambinone synthesis by the strain RK5 (+58%) compared with fermentation in a culture medium without glutamate, equivalent to strain RK4 under the same conditions. The increases observed equally approach the values of the first series of experiments.

The production of frambinone by strain RK8, cultivated without glutamate, was compared to strain RK5 under the same conditions. A +36% increase in the synthesis of frambinone, around the same observed for strains RK4 and RK5 cultivated with glutamate, was observed.

Determination of the levels of synthesis of frambinone by strains RK4, RK5 and RK8 by using the ACE_29 method similarly shows an increase in the production of frambinone, in proportions, however in higher proportions, that is, +100%, +95% and +129%, respectively.

TABLE 7 Determination by HPLC of the frambinone concentration at the end of fermentation by various strains built into the mediums without glutamate (Glu−) and with glutamate (Glu+). Frambinone concentration Long method ACE_29 (mg/L) Glu− MD Glu+ MD Increase Glu− MD Glu+ MD Increase RK4 4.60 0.32 7.23 0.18 +57% 1.09 0.17 2.17 0.02 +100% RK5 4.57 0.27 7.23 0.21 +58% 1.14 0.02 2.21 0.03  +95% RK8 6.20 0.85 n.d. n.d. +36% 2.60 0.29 n.d. n.d. +129% n.d.: undetermined MD: Standard Deviation (SD)

The results obtained using the “long method” from fermentation in a culture medium containing glutamate show an increase in the frambinone synthesis by the strain RK5 (−20%) compared with fermentation in a culture medium without glutamate, equivalent to strain RK4 under the same conditions. The decreases observed equally approach the values of the first series of experiments.

The production of tyrosol by strain RK8, cultivated without glutamate, was compared to strain RK5 under the same conditions. A −49% decrease in the synthesis of tyrosol, around the same observed for strains RK4 and RK5 cultivated with glutamate, was observed.

Determination of the levels of synthesis of frambinone by strains RK4, RK5 and RK8 using the ACE_29 method similarly shows a decrease in the production of tyrosol in similar proportions, that is, −19%, −19% and −52%, respectively.

TABLE 8 HPLC determination of the tyrosol concentration at the end of fermentation by various strains built into the mediums without (Glu−) and with (Glu+) glutamate. Concentration In tyrosol Long method ACE_29 (mg/L) Glu− MD Glu+ MD Decrease Glu− MD Glu+ MD Decrease RK4 158.8 0.3 128.7 2.2 −19% 184.2 4.4 149.2 2.5 −19% RK5 158.8 0.6 127.4 0.5 −20% 183.9 1.0 149.4 1.9 −19% RK8 80.3 4.3 n.d. n.d. −49% 88.3 5.9 n.d. n.d. −52% n.d.: undetermined MD: standard deviation (SD)

III/ CONCLUSIONS

In conclusion, introduction of the frambinone path as shown in FIG. 1 is sufficient to produce frambinone from tyrosine by means of S. cerevisiae. However, when tyrosine is added to the fermentation medium, the bioconversion is not very effective and leads to the production of a large quantity of by-products, essentially tyrosol as reported above. To reduce “hijacking” the substrate, the HPPDC activity involved in breaking tyrosine down into tyrosol was successfully reduced by adding an inhibitor of said enzyme activity or the inactivation of gene ARO10. As reported, this allowed the production of frambinone to be increased. Alternatively, other strategies can be used, such as the selection of a microbial strain with very low HPPDC activity, based on a natural genetic background or via the introduction of other genetic modifications such as the deletion of the genes encoding the HPPDC enzymes. 

The invention claimed is:
 1. A process for the production of frambinone comprising culturing a genetically modified fungal microorganism with a capacity to produce frambinone from tyrosine in a medium comprising tyrosine and a repressor of the path for breaking tyrosine down into tyrosol, p-hydroxyphenylacetaldehyde and/or p-hydroxyphenylacetate, and wherein the repressor is glutamate.
 2. The process for the production of frambinone according to claim 1 characterized in that the microorganism (a) belongs to the phyla chosen from among the ascomycetes or basidiomycetes, (b) includes at least one heterologous sequence encoding the enzyme 4-coumarate: CoA ligase (4CL) or benzalacetone synthase (BAS), (c) comprises at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia lyase (TAL) or benzalacetone reductase (BAR), (d) comprises at least one heterologous or supernumerary sequence encoding the enzyme phenylalanine ammonia lyase (PAL) or cinnamate 4 hydroxylase (C4H), or (e) comprises any combination of characteristics (a)-(d).
 3. The process for the production of frambinone according to claim 1 characterized in that the microorganism is the industrial strain Saccharomyces cerevisiae RK4 registered with the CNCM on Jun. 1, 2016 under number I-5101.
 4. The process for the production of frambinone according to claim 1 characterized in that the microorganism is the industrial strain Saccharomyces cerevisiae RK5 registered with the CNCM on Apr. 26, 2017 under number I-5199.
 5. The process for the production of frambinone according to claim 1 characterized in that the microorganism belongs to the genus Yarrowia, Debaryomyces, Arxula, Scheffersomyces, Geotrichum, Pichia, or Saccharomyces.
 6. The process for the production of frambinone of claim 2 characterized in that the microorganism includes at least one heterologous sequence encoding 4CL and BAS, at least one heterologous or supernumerary sequence encoding TAL and BAR, and/or at least one heterologous or supernumerary sequence encoding PAL or C4H. 